(1) Field of the Invention
The present invention relates to the use of isoflavonoids for the stimulation of vesicular-arbuscular mycorrhizal fungi (VAM) Fungi. In particular, the present invention relates to methods and compositions which use the isoflavonoids to stimulate the growth of plant materials in the presence of the VAM fungi.
(2) Prior Art
Several fungal species which are members of the family Endogonaceae (Zigomycetes) form endophytic symbiotic associations with roots of a large number of vascular plants in virtually all types of terrestrial habitats (Harley, J. L., and Smith, S. E., Mycorrhizal symbiosis. Academic Press, London, p. 483 (1983); Safir, G. R., Ecophysiology of VA mycorrhizal plants. CRC Press, Boca Raton. p. 224 (1987)). These associations are termed vesicular-arbuscular mycorrhizae (VAM) and are known to occur in at least 300,000 plant species, including most agriculturally important crops, with the exception of crucifers and a few other species. As widely known, the VAM fungi can have profound beneficial effects on plant growth, nutrition and tolerance to both abiotic and biotic stress (Powell, G. L. and Bagyaraj, D. J., VA mycorrhiza. CRC Press, Boca Raton. p. 234 (1984); Safir, G. R., Ecophysiology of VA mycorrhizal plants. CRC Press, Boca Raton. p. 224 (1987)). These benefits result from increased soil nutrient uptake, increased nodulation and biological nitrogen fixation in legumes, favored plant-water relationships, reduced disease severity, increased accumulation of plant growth promoting substances and other physiological effects (Smith, S. E. and Gianinazzi-Pearson, V., Ann. Rev. Plant Physiol. Plant Mol. Biol. 39:221-244 (1988)). Reseachers and commercial firms from all over the world have recognized the great biotechnological potential for large scale application of these fungi in agriculture and forestry (Jeffries, P., Use of mycorrhizae in agriculture. Crit. Rev. Biotechnol. 5:319-357 (1987); Powell, C. L. and Bagyaraj, D. J. ed. VA mycorrhiza. CRC Press, Boca Raton. p. 234 (1984); Safir, G. R., ed., Ecophysiology of VA mycorrhizal plants. CRC Press, Boca Raton., p. 224 (1987); and Siqueira, J. 0. and Franco, A. A., Biotechnologia do solo. MEC/ABEAS, Brasilia, p234 (1988)). The use of these fungi as plant or soil inoculants could have significant impacts on agriculture and global environmental quality by reducing fertilizer and pesticide use, by reducing crop loss due to abiotic (metal toxicity, drought, adverse temperature) and biotic (nematodes and pathogens attack) stresses. In addition, these fungi have been shown to improve the survival and early growth of out plantings and increase productivity or re-vegetation in poor and disturbed sites. In fact, the outgrowth of some transplanted forest and fruit species as well as coffee seedlings is known to be improved by a range of 50 to 8000% by proper VAM inoculation. For horticultural and arable crops the reported yield increases have ranged from 5 to 290% by VAM inoculation (Powell, C. L., and Bagyaraj, D. J., ed. VA mycorrhiza. CRC Press, Boca Raton. p234 (1984); Siqueira, J. 0. and Franco, A. A., Biotechnologia do solo. MEC/ABEAS, Brasilia. p235 (1988)).
Considering the concerns with environmental quality in the developed nations; the high fertilizer requirement of tropical soils, where agricultural expansion is expected to happen; and the fact that global phosphate deposits will potentially be exhausted in about 70 years; there is a tremendous worldwide market for VAM inoculum.
The importance of VAM fungi for plant growth in most soils has been accepted and acknowledged by soil chemists, agronomists, horticulturists, ecologists, farmers and nurseryman (Powell, C. L., and Bagyaraj, D. J., ed. VA mycorrhiza. CRC Press, Boca Raton. p234 (1984); Safir, G. R., ed., Ecophysiology of VA mycorrhizal plants. CRC Press, Boca Raton, p224 (1987)), but current inability to supply large quantities of active and effective inoculant represents the main drawback to their large scale use.
The VAM fungi are regarded as obligate biotrophs in that they have not been successfully cultivated under axenic conditions (Hepper, C. M., VAM spore germination and hyphal growth in vitro: prospects for axenic culture. In: Proc. 7th NACOM, Sylvia et al. (ed). University of Florida, Gainesville. 172-174 (1987)); Mosse, B. Some studies relating to "independent" growth of vesicular-arbuscular endophytes. Can. J. Bot. 66:2533-2540 (1988); Siqueira, et al Can. J. Microbiol. 31:965-972 ((1985)). The lack of infective VAM fungi makes intensive studies on the basic biology, ecology, host-relationship and inoculant production technology very difficult. The culture of VAM fungi in vitro has frequently been attempted, but has met with variable results (Hepper, C. M., VAM spore germination and hyphal growth in vitro: prospects for axenic culture. In: Proc. 7th NACOM, Sylvia et al. (ed). University of Florida, Gainesville p.l72-174 (1987)); Siqueira, et al. Mycologia 74:952-959 (1982); Siqueira, J. 0., et al., Can. J. Microbiol. 31:965-972 (1985)). European patent application No. EP0l72085 describes the axenic growth of VAM fungi; however, the growth factors are from a non-plant source and the VAM fungi would have limited ability to infect the plants. Viable spores of most species are readily germinated when plated on suitable media, but germination synchrony and the rate of germ tube growth in the absence of living roots are affected by several innate environmental and nutritional factors (Hepper, C. M., VAM spore germination and hyphal growth in vitro: prospects for axenic culture. In: Proc. 7th NACOM, Sylvia et al. (ed). University of Florida, Gainesville p. 172-174 (1987); Siqueira, J. 0. et al. Can. J. Microbiol. 31:965-972 (1985)). Although spores of most species have no specific nutritional requirement for either germination or hyphal growth, addition of certain factors has been shown to be beneficial, but only to a very limited extent (Siqueira, J. O., Can. J. Mirobiol. 31:965-972 (1985)). Continued hyphal growth and sporulation are only achieved in the presence of living plant roots (Becard, G. and Fortin, J. A., New Phytol. 108:211-218 (1988); Mosse, B., Can. J. Bot. 66:2533-2540 (1988)). Japanese Patent 63-87973 (1988) showing VAM fungi inoculated with potatoes and various growth accelerators and U.S. Pat. No. 4,294,037 to Mosse et al describes the growth of VAM fungi in the presence of plant roots.
For a long time root exudates have been thought to play an important role in the initiation and extent of VAM formation (Harley, J. 0., and Smith, S.E., Mycorrhizal symbiosis. Academic Press, London. p.483 (1983)). The presence of roots stimulates hyphal growth, even without physical contact (Becard, G. and Fortin, J. A., New Phytol. 108:211-218 (1988); Mosse, B. and Hepper, C. M., Physiol. Plant Pathol. 5:215-223 (1975); and Mosse, B., Can. J. Bot. 66:2533-2540 (1988)). However, the effects of either root exudates or extracts on spore germination or hyphal growth in vitro are very inconsistent (Harley, J. L. and Smith, S.E., Mycorrhizal symbiosis. Academic Press, London. p.483 (1983); Siqueira, J. 0., Cultura axenica e monoxenica dos fungos micorizicos vesiculo-arbusculares. In: Proc. II Reuniao bras. micorrizas, Sao Paulo, Sec. Meio Ambiente. p.44-70 (1987)). A recent study however, indicated that the quality rather than the quantity of root exudates is an important factor for VAM fungal growth in vitro (Elias, K.S. and Safir, G. R., Appl. Environ. Microbiol. 53:1928-1933 (1987)). The authors suggested the presence of a transient VAM growth factor in the root exudates of phosphorus deprived clover plants.
Non-VAM species such as Brassica appear not to be colonized because they lack a diffusible growth stimulant present near the roots of compatible hosts (Glenn, M. G., et al., New Phytol. 110:217-225 (1988). This agrees with the suggestion that chemical signaling must take place in the early events of VAM establishment (Elias, K. S., and Safir, G. R., Appl. Environ. Microbiol. 53:1928-1933 (1987); Becard, G. and Fortin, J. A., New Phytol. 108:211-218 (1988); Bonfate-Fasolo in Scannerini et al. (Scannerini, S., Smith, D., Bonfante-Fasolo, P. in Gianinazzi-Pearson, V. eds., Cell to cell signals in plant, animal and microbial symbiosis. Spring Verlag, Berlin. p.414 (1988)).
Low molecular weight phenolic compounds are known to play important roles in a wide variety of plant-microbe systems. In the plant-Rhizobium symbiosis, flavonoids present in root exudates act in a regulatory fashion as inducers or repressors of nod genes on the symbiotic plasmids of the bacteria (Rolfe and Gresshoff, Ann. Rev. Pl. Physiol. Pl. Mol. Biol. 39:297-319 (1988). For instance, luteolin, which induces nod ABC gene expression in R. melliloti, may occur in low concentrations in the alfalfa rhizosphere and limit nodulation and nitrogen fixation (Kapulnik, Y, et al., Plant Physiol. 84:1193-1196 (1987)). Flavonoids may also induce haustorial formation in parasitic plants (Steffens, J. C., et al. Ann. Bot. 50:1-7 (1982)); control plant microbial invasion (Bailey, J. A., ed. biology and molecular biology of plant-pathogen interactions. Spring Verlag, Berlin. p. 415 (1986); Palacios, R. and Verma, D.P.S., ed. Molecular genetics of plant-microbe interactions. APS, St. Paul. p.401 (1988); Templeton, M.D. and Lamb, D. J., Plant, Cell Environ. 11:395-401 (1988); VanEtten, H.D., Phytochemistry 15:655-659 (1976)); act as natural auxin regulators (Jacobs, M. and Rubery, P.H., Science 241:346-349 (1987)) and are known to accumulate as a response to pathogen invasion (Templeton, M.D. and Lamb, C.J., Plant, Cell Environ. 11:395-401 (1988)). Other low molecular weight phenolic compounds can induce gene expression of cytokinin biosynthesis and vir genes in pathogenic Agrobacterium (Conn, E. E., ed. Opportunities for phytochemistry in plant biotechnology. Plenum Press, New York, p.210 (1988)); Powell, G. K., et al., Mol. Plant-Microbe Interactions. 1:235-242 (1988)); regulate cyclic processes in lichens (Scannerini, S., et al., Cell to cell signals in plant, animal and microbial symbiosis. Spring Verlag, Berlin. p.414 (1988)). They can also act as allelopathic compounds and affect both VAM hyphal growth in vitro and root colonization (Wacker, T. L., et al., J. Chem. Ecol. (in press) (1989)).
The VAM fungi are obligate biotrophs. They may have lost the genetic capability for saprophytic growth (or have a depressed part of the required genome) during their long co-evolution with plants (Siqueira, J. 0., Cultura axenica e monoxenica dos fungos micorizicos vesiculo-arbusculares. In: Proc. II Reuniao bras. micorizas, Sao Paulo, Sec. Meio Ambiente. p. 44-70 (1987)), thus permitting the host plant to have complete control of the fungal life cycle through interference with the replication of fungal nuclear DNA as recently suggested (Burggraaf, A.J.P. and Beringer, J.E., New Phytol. 111:25-33 (1989)). In fact, the germination process is readily triggered after spore inbibition (Siqueira et al., Can. J. Microbiol. 31:965-972 (1985)), but at least for one species, nuclear DNA synthesis has not been found during in vitro development (Burggraaf, A.J.P. and Beringer, J.E., New Phytol. 111:25-33 (1989)). Additionally, neither appresoria nor arbuscules (haustorium-like structures) have reported to form in the absence of living roots. It seems that both continued hyphal growth and differentiation are under host control. As observed for other obligate biotrophic fungi (Hoch, H.C. and Staples, R.C., Ann. Rev. Phytopathol. 25:231-247 (1987)) plant messengers or inducers may be needed as triggers for the invaders early growth, development and differentiation.